Rabbit heart lipoprotein lipase (LPL) was partially purified by affinity chromatography. The purified enzyme was characterized by salt inhibition, the requirement of a serum co-factor and an alkaline pH optimum. Because of the known hypocholesterolemic effect of dietary polyunsaturated fat in this and other species, the fatty acyl substrate specificity of this lipase was also studied. Both saturated and unsaturated fatty acid chain hydrolysis were investigated using synacyl and mixed acyl triglyceride emulsion substrates. It was found that trans, monounsaturated and some saturated fatty acids were more favorably hydrolyzed than polyunsaturated cis fatty acids. Positional specificity was also observed. The physiological significance of these findings may relate to a) the production of lipoprotein remnant particles relatively enriched in polyunsaturated fatty acids, especially when consumed in the diet and b) the subsequent preferential delivery of these fatty acids to the liver.